Effect of ExSpe U1 sm21 in SMN2 Transgenic Mice
Four different SMN2 transgenic mice (Jackson Laboratories, stock 005024) of both sexes were injected intraperitonelly at P1 with scAAV9-Exspe U1 sm21 (animals 1–4), four were injected with scAAV9-U1 WT, and four animals were not treated and have been used as controls. Mice were sacrificed 4 weeks after AAV administration. Organs and tissues including brain (A), heart (B), kidney (C), liver (D), and skeletal muscle (E) were harvested and total RNA was extracted with Trizol reagent. Left: semiquantitative RT-PCR was carried out with a set of SMN2-specific primers. The resulting amplified products were solved on 1.5% agarose gels. Lanes 1 and 2 show the pattern of splicing of uninjected (CNT) and scAAV9-U1WT-injected mice, as well as the percentage of human exon 7 (hE7) inclusion expressed as mean ± SD of the four different mice belonging to each group. Lanes 3–6 show the single pattern of splicing of hE7 of the four animals (mouse 1–4) injected with scAAV9 ExSpe U1 sm21. The identity of the bands is depicted on the right of the gels and the percentage of exon inclusion for each lane, quantified by ImageJ software, is reported. Right: human-specific SMN TaqMan assay to detect the levels of E7 or ΔE7 isoforms. The levels of E7 or ΔE7 in the untreated SMN2 transgenic mouse (group 1, control) were set to 1. The histograms express the fold changes of hSMN2 E7 or ΔE7 isoforms in group 2 and group 3 animals (treated with U1 WT or ExSpe U1 sm21, respectively) compared to the untreated control group. Error bars show standard deviations (∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.1; ns, not significant. One-way ANOVA test).